Genetic and biochemical properties of apples that
affect storability and nutritional value
Masoud Ahmadi-Afzadi
Introductory Paper at the Faculty of Landscape Planning, Horticulture and
Agricultural Science, 2012: 1
Swedish University of Agricultural Sciences
Balsgård, January 2012
ISSN 1654-3580
Genetic and biochemical properties of apples that
affect storability and nutritional value
Masoud Ahmadi-Afzadi
Introductory Paper at the Faculty of Landscape Planning, Horticulture and
Agricultural Science 2012: 1
Swedish University of Agricultural Sciences
Balsgård, January 2012
2
Summary
Apple is a highly appreciated fruit in many temperate parts of the world, and is presently
grown in many countries with a total world production of more than 71 million tonnes.
Economically, apple is the fourth most important fruit crop after citrus, grapes and banana.
Apples are consumed fresh, directly after harvest or after a storage period for up to 6
months or even longer. Apples can also be processed to produce, e.g., juice, sauce, slices,
vinegar and cider. Most of the cultivated apples belong to the species Malus × domestica
Borkh. in the Rosaceae family. More than 7500 apple cultivars have been described from
different countries. However, only a few of them have sufficient quality and productivity.
Many cultivars are limited by different diseases that reduce the apple quality and market
acceptability. Research attempts have recently been focused specifically on some traits
which are economically very important, e.g. disease tolerance, fruit texture and quality.
This introductory paper forms part of a PhD study that aims to quantify the storage disease
tolerance of some apple cultivars by performing inoculation tests with fungal spores on
harvested fruits. Using DNA analysis, attempts will subsequently be made to develop tools
for molecular identification and characterization of genes involved in storage disease in cooperation with INRA Angers, using microarray technique. Other factors related to fruit
quality and nutritional value that may be connected to the level of fungal disease tolerance
will also be investigated by pomological characterization, firmness testing, and chemical
analyses.
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List of contents
Summary ....................................................................................................................................................... 3
1. Introduction .............................................................................................................................................. 6
2. Taxonomy of apple.................................................................................................................................... 7
3. Origin of apple........................................................................................................................................... 9
4. Apple cultivars......................................................................................................................................... 10
5. Biology of apple....................................................................................................................................... 12
6. Apple production and geographical distribution .................................................................................... 13
7. Apple diseases......................................................................................................................................... 15
7.1. Fungal diseases ................................................................................................................................ 15
7.1.1. Fungal diseases affect apple storage ability and fruit quality................................................... 16
7.2. Other diseases caused by bacteria and viruses ............................................................................... 17
8. Apple fruit quality and disease resistance .............................................................................................. 19
8.1. Apple contents involved in fruit quality ........................................................................................... 19
8.2. Apple chemical contents and disease resistance ............................................................................ 19
8.3. Impact of apple peel in disease resistance ...................................................................................... 20
8.4. Fruit texture, harvest time and disease resistance .......................................................................... 20
9. Breeding and biotechnology of apple ..................................................................................................... 21
9.1. Traditional apple breeding ............................................................................................................... 21
9.2. Application of molecular markers in apple ...................................................................................... 22
9.2.1. Isoenzyme markers ................................................................................................................... 22
9.2.2. DNA markers ............................................................................................................................. 23
(a) DNA markers and apple diversity .............................................................................................. 23
(b) DNA markers and gene tagging ................................................................................................. 23
(c) Marker Assisted Selection (MAS) and QTL mapping ................................................................. 24
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(d) Identifying candidate genes ...................................................................................................... 25
10. Towards a better understanding and control of fungal storage diseases in apple .............................. 26
10.1. Breeding for disease resistance ..................................................................................................... 26
10.2. Bio-control of disease resistance ................................................................................................... 26
11. Aims of this PhD study .......................................................................................................................... 28
12. References ............................................................................................................................................ 29
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1. Introduction
Apple (Malus × domestica Borkh.) is the fourth most important fruit crop after citrus,
grapes and banana, and one of the commercially most important horticultural crops grown
in temperate parts of the world (O’Rourke, 2003). Apple belongs to the Rosaceae family
which includes many well-known genera with economically important fruits, particularly
edible, temperate-zone fruits and berries such as apple, pear, almond, apricot, cherries,
peach, plums, strawberries and raspberries. Among these, apple with a world production of
more than 71 million tonnes, cultivated in many countries in the world, can be considered
as one of the most important horticultural plants (FAO, 2009).
Apple fruit has multiple uses and this fact makes it popular in the entire world, also
in areas where it is more difficult to grow. In most cases, apples are consumed fresh or
after storage for up to 6 months or even longer (usually requiring ultra-low oxygen storage
facilities). Apples can also be processed into juice, sauce, slices, vinegar and cider (Folta and
Gardiner, 2009). Apple has been considered as a symbol for the healthy fruit which
eliminates the need for a doctor: “an apple a day keeps the doctor away”.
Since a long time back, the apple tree has been cultivated and used to feed humans
and animals. Cultivation of apples has been known for 3000 years in Greece and Persia. The
Old Silk Road from eastern China to the Black Sea is claimed to have played an important
role in the evolution of cultivated apples (Juniper et al., 1999). Apples can be grown under
different climatic conditions, ranging from temperate climates such as southern Siberia or
the Mediterranean to subtropical climates such as Brazil or South Africa. Nowadays, it has
become increasingly popular to cultivate apples also in subtropical and tropical (high
altitudes) areas since they fetch a comparatively high price on the market.
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2. Taxonomy of apple
Most of the cultivated apples belong to Malus × domestica (also known as M. pumila)
in Rosaceae family. The commercial apple is a hybrid species with a complex history of
inter- and intraspecific hybridization. The scientific name of domestic apple is therefore
often written with a ‘×’ between the genus and species (Korban and Skirvin, 1984).
Rosaceae family includes many well-known and appreciated genera with economically
important edible temperate-zone fruits. Rosaceae is subdivided into several subfamilies
including Maloideae. This subfamily includes approximately 1000 species in 30 genera
characterized by the distinctive fruit, the pome, and a base chromosome number; x = 17
(Evans and Campbell, 2002; Luby, 2003; Folta and Gardiner, 2009). Maloideae contains
many of the commercially most valuable fruits like apples and pears, some ornamentals
and also invasive plants. Different studies including cytology and morphology, flavone
analysis and isozyme analysis, have suggested that Maloideae subfamily originates from
hybridization between a Spiraeoideae ancestor and a Prunoideae ancestor, followed by
fusion of unreduced gametes to form a fertile organism (Currie, 2000).
The genus Malus consists of five sections (Malus, Sorbomalus, Chloromeles, Eriolobus
and Docyniopsis) based on morphological traits and flavonoid similarities. Section Malus
consists of series Malus, including many European and Asian species (including M. sieversii
and M. × domestica) and series Baccatae. Section Sorbomalus includes series Sieboldianae
(native to Japan), Florentinae (from south-east Europe), Kansuenses and Yunnanenses.
Section Chromeles consists exclusively of North American species. Section Eriolobus
consists of only one species from eastern Mediterranean and finally, section Docyniopsis
includes some species originally from Japan, Taiwan and South-East Asia (Phipps et al.,
1990).
The total number of species in the genus Malus varies between different studies and
according to the different views on taxonomy. The maximum number of species is reported
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as many as 78 species within the genus (Robinson et al., 2001). Harris et al. (2002)
recognized 55 species while Zhou (1999) recognized only 30–35 species. According to
Robinson et al. (2001), the genus Malus comprises 25–47 species, depending on the rank
given to several taxa and the acceptance of new species and putative hybrids. The difficulty
in species delimitation has been reported to stem from the high genetic diversity,
hybridization potential, polyploidy occurrence and presence of apomixis (Campbell et al.,
1991). Morphological studies (Phipps et al., 1990; Robinson et al., 2001) along with
biochemical analysis (William, 2008) and molecular techniques have been conducted in
order to characterize and classify different species in the Maloideae subfamily.
The majority of apple cultivars are diploid with 2n = 34 and a genome of moderate
size (1C = 2.25 pg which corresponds to approximately 1.5 × 109 bp) (Janssen et al., 2008)
whereas some cultivars are triploid with 2n = 3x = 51 (Pereira-Lorenzo, 2009). Possibly,
the Maloideae subfamily has resulted from an ancient autopolyploidization of a 9
chromosome progenitor to 18 chromosomes. Then it was followed by a chromosome loss
resulting in current 17-chromosome apple cultivars whereas other subfamilies are x = 7, 8
or 9 (Folta and Gardiner, 2009; Giovannoni, 2010).
8
3. Origin of apple
Apple species are distributed throughout very large regions of the world including
West Asia, Himalayan, Central Asia, India, Western provinces of China, Europe and some
parts of America and Africa (Juniper et al., 1999). Historical studies have shown that apple
seed transfer by human or animals have probably helped in its distribution from the center
of origin (the region where the species originated) to other parts of the world.
Central Asia has been reported to contain the greatest diversity of Malus, and this
area also appears to be the center of origin of the domesticated apple (Janick et al., 1996).
This is in accordance with Vavilov’s hypothesis about the wild apples in central Asia and
their close relatives being the progenitors of the domesticated apple (Harris et al., 2002).
Nowadays, M. sieversii which grows wild in Kazakhstan and Kyrgyzstan, is thought to be
the main progenitor species (Pereira-Lorenzo, 2009). Malus sieversii has very high
similarity with M. × domestica in morphology and fruit flavor. According to observations
made on extensive collection tours, M. sieversii has been claimed to incorporate all the fruit
qualities which are present in the domesticated apples (Forsline, 1995).
Relationships among apple species have been evaluated by morphological and
molecular DNA analysis, and have confirmed that M. sieversii is the species from which
apple domestication started (Forte et al., 2002). This species may have hybridized with
M. prunifolia, M. baccata and M. sieboldi to the East and with M. turkmenorum and
M. sylvestris to the West. Subsequently, well-established apple cultivars were selected and
introduced into Europe and especially the Mediterranean regions by the Romans (Juniper
et al. 1999). Other Malus species are occasionally used for introgression into modern
cultivars but this usually requires several generations of back-crossing to reach acceptable
fruit size and quality.
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4. Apple cultivars
Malus × domestica contains over 7500 cultivars that have originated from different
countries in the world. Many cultivars have desirable characteristics which make them
suitable for cultivation under specific conditions, but only a few dozen of these are grown
commercially on a worldwide scale (Moore et al., 1991).
Development of new apple cultivars is a time- and money-consuming process since a
cultivar must be very good in several characteristics, e.g., uniform and consistent yield,
commercial fruit quality, good post-harvest storability and shipping quality, high consumer
demand and finally resistance against diseases, pests and storage disorders (O’Rourke,
2003). Recently, research attempts have become more focused on those traits which are
demanded from costumers. According to Pereira-Lorenzo (2009), important traits to
consider are fruit size, shape, color, acidity, sweetness, flavour, resistance to diseases and
abiotic stress, harvest time, storability and shelf-life. In the early and middle parts of the
last century, many small apple growers grew a few cultivars for their own use and for the
local markets. Nowadays, almost all commercially grown apples are stored for some times
before being sold, and therefore cultivars need to have good storage ability (Ferguson and
Boyd 2002). Most (old) apple cultivars have been selected in or around established
orchards as chance seedlings whereas more recent cultivars generally are derived from
breeding programs or have been selected as sports (mutants) from other cultivars (Janick
et al., 1996; Brown and Maloney., 2005). Although transgenic plants have been produced
for a number of apple cultivars (Seong et al., 2005; Chevreau et al., 2011: Wu et al., 2011),
GMO fruits have not yet been released on the market.
Some of the major important cultivars are listed in Table 1 (O’Rourke, 2003;
Hampson et al., 2003). About twenty years ago, ‘Golden Delicious’ was the most widely
grown cultivar in the world, followed by ‘Delicious’, ‘Cox’s Orange Pippin’, ‘Rome Beauty’,
‘Belle de Boskoop’, ‘Granny Smith’, ‘Jonathan’ and ‘McIntosh’ (Moore et al., 1991). Since
10
then cultivars like ‘Elstar’, ‘Fuji’, ‘Gala’ and ‘Jonagold’ have become very popular while
especially culinary apples like ‘Rome Beauty’ and ‘Belle de Boskoop’ have decreased in
popularity.
Table 1. Some important apple cultivars, country of origin and storage ability.
Cultivar
Origin
Storage ability
‘Golden Delicious’
USA
Resistant to storage disease*
‘Delicious’
USA
Medium resistant to storage disease
‘Cox’s Orange Pippin’
England
Susceptible to bitter rot, not suitable for long
storing
‘Granny Smith’
Australia
Long-keeping apple with low ethylene production
‘Jonathan’
USA
Resistant to storage disease
‘McIntosh’
Canada
Medium susceptible to storage disease
‘Jonagold’
USA
Long storability if harvested at optimal time
‘Braeburn’
New Zealand
Susceptible to bitter rot and other calcium-related
disorders
‘Elstar’
Netherland
Slightly susceptible to storage disease
‘Fuji’
Japan
Long shelf life, resistance to bitter rot
‘Gala’
New Zealand
No significant storage disease
‘Aroma’
Sweden
Susceptible to fungal decay and bruising
‘Ingrid Marie’
Denmark
Susceptible to fungal decay, cracks, and bruising
Sources: Moore et al., 1991; Hampson et al., 2003; Tahir, 2006; Ahmadi-Afzadi et al.; 2011.
* Refers especially to two major storage diseases; blue mould and bitter rot (unpublished data;
Ahmadi-Afzadi et al.).
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5. Biology of apple
The apple tree has hermaphroditic flowers with a gametophytic type of selfincompatibility controlled by a single multiallelic locus (Pereira-Lorenzo, 2009). Therefore,
at least two different cultivars must be interplanted in the orchard to ensure high levels of
cross-pollination in order to achieve adequate fruit development. Alternatively, trees of
undomesticated Malus species can be interplanted among (or top-worked onto) some of
the trees in the row. Blooming and ripening time of different cultivars vary considerably
and form different categories, e.g. early, middle and late blooming or ripening cultivars.
Flowering in apple is the result of several physiological changes from vegetative to
reproductive phase. Like in many other fruit crops, newly initiated apple buds become
dormant in late summer or early autumn. Winter chilling (defined as a certain number of
hours at or below 7.2 °C) is necessary to break bud dormancy. If chilling is not sufficient,
both flower buds and vegetative buds (producing leaves only) are delayed. Flowering time
of the different cultivars must overlap to a large extent for pollination to be successful.
Number of days from pollination to fruit ripening varies considerably due to inherent
differences between cultivars and to environmental effects (e.g., weather conditions).
During this period, physiological processes like cell division and expansion, starch
accumulation, ethylene production, and color changes take place (Janssen et al., 2008).
12
6. Apple production and geographical distribution
The global apple production remained stable for a large part of the previous century
until China began to expand its apple production in the 1990s. Currently, China is the
largest apple producer in the world with a production of more than 31 million tonnes,
which is several times higher than the production of the four countries in the closest
positions, e.g. USA, Turkey, Poland and Iran. China is currently responsible for
approximately half of the world apple productions. Some of the main apple-producing
countries and their production volumes are listed in Table 2 (FAO, 2009).
Most temperate-zone woody deciduous trees, including apple, require a certain
amount of chilling accumulation during the wintertime to break bud endodormancy before
active shoot growth in the spring and for normal growth (O’Rourke, 2003). In general,
apples are therefore suitable for growing mainly in areas with a temperate climate.
However apples can also be grown in other climates, like subtropical and even tropical
areas at high altitudes, where sometimes two crops can be produced per year (PereiraLorenzo, 2009). Apple production has thus been reported from countries like India, Mexico,
Brazil, Egypt, South Africa, Kenya, Ethiopia, Uganda and Zimbabwe (Wamocho and
Ombwara, 2001; Ashebir et al., 2010). In the subtropical and tropical areas of Asia, India
appears to be the largest apple producer. Many different apple cultivars are grown in the
northern, mountainous parts of the country, especially in the provinces of Jamma and
Kashmir (Verma et al., 2010). In the subtropical areas of the America, apples are grown,
e.g., in the highlands of the northern regions of Mexico and also in large subtropical areas of
Brazil (Leite, 2008; Hauagge, 2010). In Africa, the most important apple-growing country is
South Africa, where roughly 20,000 ha of apple are cultivated (Cook, 2010).
13
Table 2. List of main apple producing countries in the world (FAO, 2009)
Country
Production (MT*)
Country
Production (MT)
China
31684445
Syria
360978
USA
4514880
New Zealand
357000
Turkey
2782370
Belgium
310000
Poland
2626270
Australia
291134
Iran
2431990
Serbia
281868
Italy
2313600
Portugal
280078
France
1953600
Algeria
267496
India
1795200
Switzerland
252086
Russia
1596000
United Kingdom
243000
Brazil
1222890
Greece
235000
Chile
1090000
Moldova
210000
Germany
1070680
Azerbaijan
204237
Argentina
1027090
Czech Republic
170400
Japan
845600
Tajikistan
148000
Ukraine
853400
Kyrgyzstan
146000
North Korea
719682
Peru
137044
South Africa
702284
Lebanon
126500
Uzbekistan
635000
Israel
114378
Spain
594800
Kazakhstan
112000
Hungary
575368
Armenia
110000
Egypt
550000
Tunisia
110000
Mexico
525000
Macedonia
106356
Romania
517491
Slovenia
95662
Austria
485609
Georgia
80700
South Korea
480000
Afghanistan
72765
Belarus
431573
Bosnia and Herzegovina
71507
Canada
413096
Turkmenistan
64000
Netherlands
407000
Uruguay
58775
Morocco
400000
Lithuania
53259
Pakistan
366360
World production
71286632
* million tonnes
14
7. Apple diseases
Apples are subjected to a variety of diseases with several causal agents e.g. fungi,
bacteria, viruses, mycoplasmas and nematodes but there are also disorders with unknown
causal agents. Most disorders result in the loss of total yield. The economic losses caused by
different diseases can be exceedingly variable according to the pathogen vigor, i.e., some
are able to kill whole tree, others can infect fruits and make them unmarketable whereas
others may cause only minor symptoms. Disease control is a major annual expense for
growers in most apple-producing areas. The grower needs to control early-season diseases
like apple scab as well as summer diseases and also some storage diseases. A wellintegrated approach is usually needed to achieve successful disease management, e.g.,
application of fungicides, pesticides and bactericides (the latter usually not allowed in
Europe), selection of resistant or tolerant rootstocks and scion varieties, biological disease
control and selection of a suitable site for the orchard (Jönsson, 2007; Dewasish and Amal,
2010). Some of the most important apple diseases are described below.
7.1. Fungal diseases
Fungal diseases are the main problem for commercial apple production in humid
regions. It has been reported that apple is host to over 70 infectious diseases which most of
them are caused by pathogenic fungi. They cause root rots, leaf spots, leaf blights, blossom
blights, fruit decay, fruit spots, canker and post-harvest decay. Apple scab (Venturia
inaequalis) is usually the main apple fungal disease in commercial apple production in
temperate and humid regions. Scab mainly attacks the leaves and fruits (Sandskär, 2003).
Apple cultivars differ greatly in regard to their resistance level to scab. In Europe and New
Zealand, over 50 scab-resistant cultivars have been introduced based on apple breeding
programs, e.g. ‘Prima’, ‘Redfree’ and ‘Liberty’ (Benaouf and Parisi, 2000; Bowen et al.,
2011).
15
Powdery mildew (Podosphaera leucotricha) can be a serious disease wherever apples
are cultivated. It usually infects leaves, flowers and even fruits with masses of fungal
mycelia and spores spread over the surface. Powdery mildew distribution and epidemic is
strongly dependent on environmental conditions, e.g., relative humidity, hourly ambient
temperature and total daily duration of rainfall. In order to control powdery mildew,
application of fungicides is recommended (Moore et al., 1991; Grove et al., 2003).
Brown-rot caused by Monilinia fructicola is another apple disease which causes
blossom wilt, spur dieback, cankering and fruit rot. This disease is usually more
problematic around harvest time because commercial losses due to fruit decay increase
gradually up to harvest time and it is associated to numbers of injured fruits (Grove et al.,
2003). Black rot caused by Botryosphaeris obtusa, Sooty blotch caused by Peltaster
fructicola, Brooks fruit spot caused by Mycosphaerella pomi, Crown and root rot caused by
Phytophthora spp., and European canker caused by Nectria galligena are also important
apple diseases (Xu and Robinson, 2010).
7.1.1. Fungal diseases affect apple storage ability and fruit quality
Blue mould caused by Penicillium expansum is the most common post-harvest
disease of apple fruits. It mainly attacks injured and physically damaged fruits and
produces soft, malodorous lesions with a dark brown color. The fungus then proceeds to
produce green to blue conidia on the fruit surface. The quick spread of the disease during
storage causes much infection and subsequent severe fruit loss in commercial apple
production (Rosenberger, 1990; McCallum et al., 2002; Pianzzola et al., 2004).
Meanwhile, Penicillium produces the carcinogenic mycotoxin patulin in decayed
fruits. This mycotoxin is a major health hazard for people who consume high quantities of
fruit juices (Brause et al., 1996; Beretta et al., 2000). In order to eliminate the damaging
effect of P. expansum during storage and to avoid health problems, some efforts have been
16
made to identify and introduce tolerant cultivars (Pianzzola et al., 2004; Moake et al.,
2005).
Bitter rot is known as one of the most destructive and difficult to control apple
diseases when an epidemic has occurred. It is caused by Colletotrichum gloeosporioides and
Colletotrichum acutatum. This disease usually begins by release of conidia and infection of
the fruits in late spring when temperatures become higher. The lesions on the fruits are
small, circular, light tan to brown spots in the beginning, and then become larger and more
brown. This disease can also be considered as a problematic post-harvest disease in many
commercial apple orchards (Peres et al., 2005; Jönsson, 2007).
Another important storage disease is Bull’s eye rot caused by Pezicula malicorticis.
Infection can occur at any time during fruit development until the harvest, but usually does
not become visible on the fruits until later when exposed to cold-storage temperature,
during transport and in the shops. The lesions are most often brown with a pale center that
looks like a bull's eye (Tahir, 2006; Valdebenito et al., 2010).
Moldy-core and core rot are also other important apple fruits diseases which cause
production losses during fruit ripening and storage. Moldy core decay is predominantly
caused by Alternaria alternate and wet core rot caused by Penicillium spp is typically found
after harvest when fruits are in storage (Turechek, 2004).
7.2. Other diseases caused by bacteria and viruses
Fire blight, caused by Erwinia amylovora, also known as fruit blight, pear blight and
spur blight, is a very serious bacterial disease which affects tree trunks and branches, and
can kill a whole orchard in only a few years. It has been reported on more than 200 species
of plants whereas the main host species are in Rosaceae family. It is a common disease in
warm and temperate regions. Apple cultivars are widely different in their resistance to fire
blight. ‘Rome Beauty’, ‘Jonathan’ and ‘Granny Smith’ are susceptible whereas ‘Delicious’,
‘McIntosh’ and ‘Golden Delicious’ are resistant (Moore et al., 1991; Khan et al., 2006;
17
Nybom et al., in press). Viruses, viroids, phytoplasma and other virus-like organisms
produce over 50 identified diseases in apple. They are widely different in their
destructiveness. Some of the important diseases are apple mosaic, flat limb, tomato ring
spot and chlorotic leaf spot with symptoms on several parts of the tree (Ram and Bhardwaj,
2004).
18
8. Apple fruit quality and disease resistance
8.1. Apple contents involved in fruit quality
Fruit quality is defined as degree of excellence of fresh fruits and it is a combination
of different characteristics or properties. These characteristics are usually attractive to
consumers in terms of market acceptability or human health improvement (Kader, 1999).
It has been reported that consumers generally prefer apple fruits that are juicy, crisp and
sweet. Meanwhile, there are several other factors that determine the fruit quality and some
of these may be associated to disease resistance, e.g., hardness, acidity, ethylene production
level, flesh texture, antioxidant content, phenol content, harvest time and fruit maturity
(Jenks and Bebeli, 2011; Nybom et al., in press). Variation in phytochemical content is
caused by many factors, such as heritable traits of the cultivars, harvest and storage
procedures, and processing of the apples (Boyer and Liu, 2004).
8.2. Apple chemical contents and disease resistance
As already mentioned, different compounds in the fruit can probably play a role in
resistance to fungal diseases, especially storage diseases. It has been reported that total
phenol content is one of the factors affecting the apple storability and disease resistance. A
large number of volatile compounds are important in disease resistance of apple like
alcohols, aldehydes, carboxylic esters and ketones. In resistant cultivars, phenolic
components accumulate at a higher rate than in susceptible cultivars (Dixon and Hewett,
2000; Usenik et al., 2004; Treutter, 2005; Lattanzio et al., 2006). Among phenolic
compounds, the flavonoid quercetin has been considered the most important agent.
Sanzani et al. (2009a) has recently investigated the role of quercetin as an alternative
strategy to control blue mould and patulin accumulation in ‘Golden Delicious’. By
exogenous application of different phenolic compounds, they found that quercetin is
effective in controlling blue mould and patulin accumulation. Subsequent studies have
demonstrated that this control is achieved through an increased transcription of genes
19
involved in the quercetin biosynthetic pathway (Sanzani et al., 2009b; Sanzani et al., 2010).
In a wide variety of plants, organic acids and nutritional compounds such as vitamin
C and glutathione are associated with fruit taste and quality. These compounds are
apparently related also to the level of disease resistance (Ferguson and Boyd, 2002). The
relationship between harvest day and vitamin C content of apple fruits has been
investigated by Davey et al. (2007). Low pH can enhance P. expansum colonization, which
means that cultivars with a lower pH in their fruits are more susceptible to fungal attack
(Prusky et al., 2004).
8.3. Impact of apple peel in disease resistance
It has been hypothesized that the main defense mechanism against fungal infection
involves the fruit peel. According to many authors, most of the apple phytochemicals such
as ascorbic acid, glutathione, antioxidative enzymes, phenols and cuticular waxes like
ursolic acid, are mainly localized in the peel. Ursolic acid is a ubiquitous triterpenoid and
the main cuticular waxes present in apple peel that can be considered as a post harvesting
parameter in order to reduce shelf life diseases (McGhie et al., 2005; Frighetto, et al., 2008).
An environmental impact has also been shown. Thus, the sun-exposed side of the apple
contains a higher level of antioxidants that are involved in resistance to decay caused by
fungi (Ma and Cheng, 2003).
8.4. Fruit texture, harvest time and disease resistance
The association between fruit quality (firmness and softening), harvest date and level
of resistance to post harvest diseases has been investigated (Ahmadi-Afzadi et al., 2011;
Kellerhals et al., in press). A significant difference was noted between investigated cultivars
regarding the size of disease symptoms resulting from inoculations. Late-ripening cultivars
with high levels of firmness and little softening were, as expected, the least affected by blue
mould. From a genetic point of view, the most interesting cultivars are, however, those that
had relatively small symptoms in spite of being early-ripening and/or only medium firm
(Ahmadi-Afzadi et al., 2011).
20
9. Breeding and biotechnology of apple
Apple production in the world, presently around 71.7 million tonnes (FAO), suffers
great losses every year due to different diseases during growth season, during harvest and
also during post-harvest processing. To reduce the production losses, improvement of
disease resistance is one of the most important steps. Presently, breeders have focused
more on resistance when developing new cultivars. Breeding of disease resistant cultivars
can also reduce disease control costs and meet consumer demands concerning the
avoidance of pesticide residues in the fruits. Identification and breeding of such cultivars
will increase the level of disease tolerance in the field. A main step in breeding is to gain
better knowledge about genetic resources that are suitable in breeding programs, and this
will also help to conserve biological diversity.
9.1. Traditional apple breeding
Records of human use of apples originate from the beginning of civilization when
agriculture and apple growing was initiated. The earliest application of apple breeding took
place when humans simply selected nice apples from different trees. Selection based on
desirable traits can thus be seen as the first step of breeding. The apple breeding process
was also influenced by the invention of grafting. Morgan et al. (1993) reported that grafting
genotypes would have increased the quality of apple orchards because only the best
cultivars would have been propagated rather than a random collection of their offspring.
By introduction of controlled pollination systems and development of new crossing
techniques, breeders focused more on breeding based on crossing to produce seeds with a
known pedigree. The first controlled pollination apple breeding program was done when
Thomas A. Knight (1806) crossed different apple genotypes and then selected superior
phenotypes. This is still the way in which breeders conduct the breeding process; mating
parents with suitable traits in order to transfer a desirable trait in the pollen parent to a
recipient seed parent with a superior phenotype. It is the most effective way to increase the
21
frequency of the desirable alleles due to the relatively high additive variance in most of the
traits (Janick et al., 1996; Folta and Gardiner, 2009). Another commonly used strategy is
mass selection. In this strategy, apple breeders select parents from commercial cultivars
with favorable characteristics, cross them and then select progeny to test on rootstock for
commercial release (Janick et al., 1996).
9.2. Application of molecular markers in apple
Traditional breeding of new valuable apple cultivars takes a long time and is very
costly in most cases. Therefore, the efficiency of apple breeding can be enhanced by use of
more informative and precise techniques such as molecular markers. Molecular markers
can be used for different purposes; some are just used for generating genotype-specific
DNA profiles while others are used to tag genes and thus help to, e.g., select desirable
seedlings.
Reliable and reproducible markers linked to desirable traits can be applied in
breeding programs. Molecular markers can help the breeder to choose better parents for
the crosses and reduce the time needed for making selections among the seedling offspring,
and consequently increase the breeding efficiency.
Molecular markers are classified into different categories, e.g., biochemical i.e.
isoenzyme and DNA markers. They can be biomolecules related to a genetic trait, or just a
difference in the sequence of a piece of DNA.
9.2.1. Isoenzyme markers
Isoenzymes are different forms of an enzyme that vary in size or conformation.
Isoenzyme markers have been used for clonal identification of apple (Gardiner et al., 1996),
and for developing markers for important genes (Hemmat et al., 1994; Chevreau et al.,
1999; Pereira-Lorenzo et al., 2003). Presently their role has, however, been overtaken by
DNA-based markers.
22
9.2.2. DNA markers
Because of the low level of polymorphism in isoenzymes, other groups of molecular
markers, i.e. DNA markers, were developed that are able to detect more polymorphism.
DNA markers are classified into a wide range of different discriminative techniques that
reveal the genetic diversity between or within different species or cultivars. DNA markers
are widely used for various purposes like studies of genetic diversity and phylogenetic
analyses (Coart et al., 2003), constructing linkage maps (Liebhard et al., 2002), QTL
analysis (Liebhard et al., 2003) and marker assisted selection (Costa et al., 2004).
(a) DNA markers and apple diversity
Different types of DNA markers have been used to evaluate the genetic diversity of
apple cultivars. Goulao and Oliveira (2001) evaluated the degree of similarity between 41
commercial cultivars of apple with 13 SSRs (simple sequence repeats) and seven ISSRs
(inter-simple sequence repeats) markers. Genetic similarity of 41 apple cultivars was
assessed by RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment
length polymorphism) markers by Goulao et al. (2001). Oraguzie et al. (2001) used RAPD
to evaluate the genetic relationships among four subsets of apple germplasm (including
155 genotypes; modern and old cultivars) in New Zealand. In other studies, genetic
diversity of apple genotypes has been evaluated with different markers like RFLP
(restriction fragment length polymorphism) (Gardiner et al., 1996a), AFLP (Tignon et al.,
2000, 2001), SSRs (Oraguzie et al., 2005; Pereira-Lorenzo et al., 2007) and RAPDs (Royo
and Itoiz, 2004).
(b) DNA markers and gene tagging
Presently, there is much research on investigation of molecular markers linked to
genes controlling apple traits. Several molecular markers associated with resistance to
apple scab have been identified. Among the major identified genes, Rvi6 (Vf) gene was the
first attractive scab resistance gene used in breeding programs around the world (Koller et
al., 1994; Manganaris et al., 1994; Hemmat et al., 1995; Gardiner et al., 1996b; Tartarini,
23
1996; Yang and Korban, 1996). However, some races of V. inaequalis have been able to
overcome the Rvi6 (Vf) resistance and started to attack formerly resistant apple cultivars in
the 1990s. A promising way to reach a more durable form of resistance can be achieved by
pyramiding genes (incorporation of two or more resistance (R) genes in the same cultivar)
(Xu and Korban, 2000; MacHardy et al., 2001). This method can delay or even prevent the
breakdown of the R genes and create cultivars with durable resistance to apple scab.
DNA based markers have been identified also for some other important genes,
including, e.g., genes related to ethylene biosynthesis and firmness of the fruits like MdACS1 (Costa et al., 2005; Oraguzie et al., 2007; Li and Yuan, 2008; Nybom et al., 2008b; Zhu
and Baritt, 2008), Md-ACO1 (Costa et al., 2005), Md-PG1 (Wakasa et al., 2006) and Md-Exp7
(Costa et al., 2008), genes related to chilling requirement (Chr) (Lawson et al., 1995), genes
related with apple fertility (MADS-box) (Yao et al., 1999), fruit color (Cheng et al., 1996),
resistance to fire blight (Malnoy et al., 2004) and powdery mildew (Markussen et al., 1995).
(c) Marker Assisted Selection (MAS) and QTL mapping
Marker Assisted Selection (MAS) is selection based on molecular markers that are
linked to a favorable trait. The target trait can either be a qualitatively inherited trait
(regulated by a monogene or major gene) or a quantitatively inherited gene (minor gene or
QTL: Qualitative Trait Locus). Because many apple traits of agronomic importance are
qualitative with clear and easily interpreted inheritance, most researches into markers
have focused on qualitative traits. As already mentioned, many DNA markers linked to
genes have already been identified. These markers can be used in different ways such as:
early selection of traits (traits can be screened during the juvenile phase), use of traits as
markers to identify the transformed plant after transformation (marker traits) and
selection of traits which are too expensive or difficult to measure directly (Currie, 2000).
Several complete or partial genetic maps based on linkage analysis have already been
developed for the apple genome. A saturated apple genome map was constructed by
24
Liebhard et al., (2003) with different types of DNA markers e.g. AFLP, SSRs, RAPD and
SCAR. Two other genetic maps have also been developed by Silfverberg-Dilworth et al.,
(2006) and Fernandez-Fernandez et al. (2008) based on just one single population.
Recently, an integrated consensus genetic map was constructed by N’Diaye et al. (2008).
This consensus map was constructed based on segregation data from four genetically
connected crosses (Discovery × TN10-8, Fiesta × Discovery, Discovery × Prima, Durello di
Forli × Fiesta) with a total of 676 individuals.
(d) Identifying candidate genes
Beside the molecular markers, other techniques are now available to analyze the
pattern of gene expression in plants. One of these techniques is the microarray technology
that can be used to identify interaction between expressed genes and a specific trait. The
DNA microarray technique is a high throughput technology by which the expression of the
whole genome is studied in a single experiment. Recently, identification of putative
candidate genes controlling fruit quality was investigated and the results showed that a
microarray could be used to identify candidate genes potentially correlated to fruit quality
QTLs (Soglio et al., 2009). Map positioning and functional allelic diversity of a new putative
expansin gene (Md-Exp7) associated with fruit softening was analyzed by Costa et al.
(2008). In another study, heterologous comparative genomics of apple was conducted to
identify candidate genes involved in fruit quality (Costa et al., 2009).
25
10. Towards a better understanding and control of fungal storage diseases in apple
10.1. Breeding for disease resistance
Postharvest decay caused by fungal diseases is the major factor limiting the storage
life of apples. Although control of these pathogens can be achieved by the application of
chemical fungicides, environmental concerns and increasing public concern about the
impact of chemicals on human health requires the development of new approaches. As
mentioned above, new high-quality apple cultivars with resistance to postharvest
pathogens can be developed via breeding methods. For some diseases like apple scab,
several sources of resistance are known (Jönsson and Tahir, 2005; Tahir and Jönsson,
2005; Nybom et al., 2008a). No major genes have as yet been described for resistance
against the common storage diseases but quantitatively inherited resistance probably
exists. Possibly, level of tolerance against storage diseases is also related to some other
traits that have already been evaluated, such as the contents of ethylene and polyphenolics
(Nybom et al., 2008b; Blazek et al., 2007) and the acidity of apple tissues (Prusky et al.,
2004). Other properties are also apparently involved in the resistance to fungal diseases in
apple, e.g., fruit firmness as well as some physiological characteristics, e.g., fruit maturity
and harvest time.
10.2. Bio-control of disease resistance
Much concern has recently been raised about patulin produced by P. expansum.
Patulin is a mycotoxin, which is harmful to human health. The application of synthetic
chemicals such as fungicides is a primary method for prevention of postharvest decay of
apple fruits in order to eliminate patulin production. However, restrictions are being made
on the use of fungicides because of the public concerns regarding human health and also
because of the environmental risks of these chemicals. Therefore, alternatives to the
conventional fungicides are needed to reduce losses from postharvest decay. Different bio-
26
control strategies can be applied to control decay caused by fungi, such as the use of
antagonistic microorganisms or natural biocides, and the increase of natural defense
mechanisms involving some plant components, such as phenolics, e.g., quercetin (Sanzani
et al., 2009a) or ARS (Dey and Mikhailopuloa, 2009).
Phenolic lipids alkylresorcinols (ARs) are secondary metabolites synthesized mainly
by plants and by a few fungi or bacteria. The synthesis can take place during normal
development and/or in response to stressful conditions such as infection, wounds, and UV
radiation. ARS have shown an inhibitory effect on bacteria, fungi and insects while they do
not show any obvious negative effect on animals or humans. Therefore, these compounds
can be applied as inhibitory agents against fungi (García et al., 1997; Hassan et al., 2007;
Dey and Mikhailopuloa, 2009).
27
11. Aims of this PhD study
This PhD study aims to quantify the storage disease tolerance of some apple cultivars
by performing inoculation tests with fungal spores on harvested fruits. Some factors that
may be connected to the level of fungal disease tolerance will also be investigated: by
pomological characterization (e.g. ripening rate), mechanical testings (e.g. fruit texture),
chemical testings (contents of chemical compounds in the fruit flesh, some of which may
also have health-promoting actions) and DNA marker screenings (e.g. ethylene-affecting
genes). Bio-control of storage disease will also be investigated by spraying of ARScontaining solutions on P. expansum-inoculated fruit of different apple cultivars. The
inhibitory effect of these ARS-containing solutions will then be evaluated by measuring the
amount of the fungal symptoms. Finally this project will also include molecular
identification and characterization of genes involved in storage disease. This will be carried
out in co-operation with INRA Angers, using apple microarrays to identify candidate genes
involved in fungal resistance and possibly also other traits like fruit softening and ethylene
production which are related to fruit resistance. Application of these markers will make it
possible to screen and classify apple cultivars regarding their disease tolerance.
28
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